Protein phosphorylation has been implicated in a number of important physiologic events ranging from normal cell growth and differentiation to malignant transformation. Phosphorylation is mediated by a superfamily of enzymes known as protein kinases. In eukaryotic cells, these enzymes phosphorylate hydroxyl groups of serine (ser), threonine (thr) and tyrosine (tyr) residues by transferring the .lambda. phosphate from ATP using a magnesium or manganese ion cofactor. Tyrosine kinases, unlike ser/thr kinases, prefer manganese ion over magnesium ion. Although the vast majority of kinases are specific to either tyr or ser/thr, several recently-described kinases can phosphorylate all three residues (Ben-David et al., (1991) EMBO J., 10: 317-325; Howell et al., (1991) Mol. Cell. Biol., 11: 568-572). These proteins are called "dual specificity protein kinases" (Lindberg et al., (1992) Trends Biochem. Sci., 17: 114-119).
Tyrosine phosphorylation is an early signal transduction event which occurs after the binding of growth factors, hormones or cytokines to cell surface receptors and is a mechanism by which a number of oncogene products exert their proliferation-inducing effects (Aaronson, (1991) Science, 254: 1146-1153; Hanks et al., (1988) Science, 241: 42-52; Hunter and Cooper, (1985) Annu. Rev. Biochem., 54: 897-930). Oncogenes are mutated forms of normal genes (proto-oncogenes) which have been picked up by retroviruses. Most proto-oncogenes encode proteins mediating events by which growth factors stimulate normal cell division (Cantley et al., (1991) Cell, 64: 281-302). For example, the v-src and v-abl genes encode transforming tyrosine kinases from Rous sarcoma virus and Abelson murine leukemia virus, respectively, which are oncogenic counterparts of the corresponding normal cellular genes. These kinases may be classified as either receptor (v-erb B, v-neu) or nonreceptor (v-src, v-abl) tyrosine kinases.
Changes in tyrosine phosphorylation are believed to be responsible for initiating reaction cascades leading to the covalent modification of proteins. In some instances, these other proteins are kinases. Many of these proteins, including p34.sup.cdc2, and the platelet derived growth factor receptor, are either protein kinases or are regulated by their phosphorylation state. Since several protein kinases, including p34.sup.cac2, regulate entry and progression through the cell cycle, they thus represent important control points in cell growth.
To elucidate the mechanisms involved in the control of cell proliferation, additional novel kinases have been identified by either low stringency hybridization and polymerase chain reaction using primers corresponding to conserved kinase regions (Kraus and Aaronson, (1991) Methods Enzymol., 200: 546-556; Wilks, (1991) Methods Enzymol., 200: 543-546) or by expression cloning (Lindberg et al., (1988) Oncogene, 3: 629-633). Enzymatically active tyrosine kinases undergo autophosphorylation on their own tyrosine residues, allowing their detection by antibodies against phosphotyrosine (pTyr). Since bacteria lack protein tyrosine kinases (PTKs), antibodies to pTyr have been particularly useful in the identification and characterization of several novel kinases isolated from bacterial expression systems (Kornbluth et al., (1988) Mol. Cell Biol., 8: 5541-5544; Letwin et al., (1988) Oncogene, 3: 621-627). Most recently, two novel protein kinases called TIK and TTK have been described (Icely et al., (1991) J. Biol. Chem., 266: 16073-16077; Mills et al., (1992) J. Biol. Chem., 267: 16000-16006). TIK is a ser/thr kinase containing consensus ser/thr kinase catalytic residues while TTK is predominantly a ser/thr kinase with a low level of tyrosine phosphorylation activity and is associated with cell proliferation.